This is a competing renewal of a grant Identification and characterization of cellular factors involved in HCV entry first funded in 2006. Our laboratory has been studying the mechanisms of hepatitis C virus (HCV) gene expression and replication for over twenty years, but our understanding of viral entry was limited by inadequate study systems. We and others overcame this hurdle by developing the first platforms for studying HCV entry in vitro - HCV pseudoparticles (HCVpp) and the fully-infectious HCV cell culture system (HCVcc). With the advent of these new tools, we proposed to identify and characterize factors required for HCV uptake. A long- term goal of these studies was to use this information to create much needed small animal models for HCV. Using lentivirus-based cyclic repackaging schemes, we succeeded in identifying two tight junction proteins, claudin-1 and occludin, as critical HCV entry factors. We built on this finding by elucidating the human factors required for viral entry into mouse cells, and have now gone on to create the first genetically humanized mouse model supporting the entire HCV lifecycle. This achievement opens unprecedented opportunities to study HCV entry in vivo and to dissect it using the power of mouse genetics. We have also shown proof-of-concept for this animal model in passive and active vaccine strategies. In addition to advancing in vivo models, we are pursuing more biologically relevant cell culture systems for the virus. These include primary adult and fetal hepatocyte cultures and three-dimensional liver organoids. In this competing renewal, we employ our novel in vitro and in vivo systems to further understand the virus-host interactions required for HCV entry. We propose genetic and biophysical approaches to uncover the mechanisms of uptake. Our studies will, for the first time, define the composition and structure of the infecting particle, probe the interplay between virus and host- associated lipid metabolism factors, and map the molecular interaction network active during viral internalization.